magnetic resonance detection: spectroscopy and imaging … · magnetic resonance detection:...
TRANSCRIPT
Magnetic resonance detection: spectroscopy and imaging of lab-on-a-chip
Elad Harel
Received 25th April 2008, Accepted 19th August 2008
First published as an Advance Article on the web 16th October 2008
DOI: 10.1039/b807036a
This mini-review is focused on the use of nuclear magnetic resonance (NMR) spectroscopy and imaging
to study processes on lab-on-a-chip devices. NMR as an analytical tool is unmatched in its impact
across nearly every area of science, from biochemistry and medicine to fundamental chemistry and
physics. The controls available to the NMR spectroscopist or imager are vast, allowing for everything
from high level structural determination of proteins in solution to detailed contrast imaging of organs
in-vivo. Unfortunately, the weak nuclear magnetic moment of the nucleus requires that a very large
number of spins be present for an inductively detectable signal, making the use of magnetic resonance
as a detection modality for microfluidic devices especially challenging. Here we present recent efforts to
combat the inherent sensitivity limitation of magnetic resonance for lab-on-a-chip applications.
Principles and examples of different approaches are presented that highlight the flexibility and
advantages of this type of detection modality.
Introduction
While optical detection is overwhelmingly the method of choice
for analysis of fluid transport in microstructured devices,1 it has
some serious limitations. Laser induced fluorescence (LIF), the
most sensitive and widely used technique is well suited for
naturally fluorescent molecules or for analytes that can be made
fluorescent through interaction with a fluor. Unfortunately, most
biomolecules and chemical compounds do not conform to this
stringent requirement. The most notable restriction of optical
methods is the need for optical access to the region of interest
which necessarily limits the range of materials that can be used
for device fabrication. While one material may work well for UV
detection, it may bode poorly for light in the visible or IR region
of the spectrum. Secondly, tracers which are typically needed to
visualize the fluid flow path can alter the hydrodynamic prop-
erties at these small dimensions.2 Another limitation of optical
detection is that the short optical path length through the device
makes absorption based spectroscopy such as UV on the chip
difficult. Other detection techniques such as IR detection3 have
been reported on microfluidic devices, although generally they
can only analyze one point on the device at a time, precluding
imaging. Confocal Raman spectroscopy does seem to be
a promising approach for spectroscopic imaging in microfluidic
devices,4 although it is typically restricted to analyzing known
compounds. Furthermore, long acquisition times do not allow
for on-line, time resolved studies needed for many kinetic
problems.
Magnetic resonance can bypass some of these limitations
because of its non-invasiveness and ability to incorporate
imaging and spectroscopy simultaneously.5 Spectroscopic
imaging in which a spectrum is acquired for every pixel in the
image is routinely practiced in medical imaging applications and
is becoming increasingly used for materials characterization.6
Furthermore, through the incorporation of multidimensional
techniques, NMR can elucidate structure and dynamics of large
molecules and proteins in solution.7 For all its prowess, however,
the sensitivity of magnetic resonance is noticeably poor
compared to most other detection techniques due to the small
energies involved even at the highest available magnetic field
strengths. This presents itself as a particular challenge in
microfluidic applications where picomolar or smaller quantities
of analyte are measured. Compounding this problem is the fact
that the radio-frequency (RF) excitation and detection must
occur over the volume of the entire chip, while only a fraction is
occupied by the fluid that gives rise to the NMR signal. For
microfluidics the direct sensitivity is less than 10!4 of traditional
high-resolution NMR under these conditions.
While several methods have been developed to deal with ultra-
small-samples such as microsolenoid RF coils for NMR,8–10 and
magnetic resonance force microscopy (MRFM),11 to name a few,
this review is concerned only with those techniques compatible
Elad Harel received his BA in
Mathematics and BS in Chem-
ical Physics from the University
of California, San Diego in
2003, under the supervision of
Robert E. Continetti. Harel then
went on to receive his PhD at the
University of California, Berke-
ley in Physical Chemistry under
the guidance of Alex Pines in the
spring of 2008. His research
focus at Berkeley was in devel-
oping novel detection methods
using NMR and MRI as it
applies to porous materials and
microfluidics. In the fall of 2008, he will start a postdoctoral
fellowship at the University of Chicago.
Department of Chemistry, University of California, Berkeley, CA, 94720,USA. E-mail: [email protected]; Tel: +1 510 642 2094
This journal is ª The Royal Society of Chemistry 2009 Lab Chip, 2009, 9, 17–23 | 17
FRONTIERS www.rsc.org/loc | Lab on a Chip
with conventional planar microfluidic devices of arbitrary
channel geometry under standard operating conditions. This
requirement limits the discussion to those methods in which any
location of the device can be analyzed either spectroscopically or
through imaging without making physical contact with the
sample such as necessary with force detection. Furthermore,
force detection requires very low temperatures not compatible
with liquid samples. Microsolenoids, while extremely sensitive,
cannot be readily integrated into the device simply due to the
incompatibility of geometries and, hence, are outside the scope of
this review.
Here we review several methods that overcome the sensitivity
limitation of traditional magnetic resonance on lab-on-a-chip.
The methods can be grouped into two categories: direct detection
and remote detection. The merits and pitfalls of each as well as
prospective avenues for future improvements are discussed.
Direct detection
Direct detection refers to the class of experiments were the
excitation and detection of the nuclear spins occurs using
the same radio-frequency coil, as in conventional NMR.12 The
signal-to-noise of an NMR experiment utilizing inductive
detection and assuming uniform sensitivity throughout the
sample is given by13
SNR ¼ gðB1=iÞVsNh2IðI þ 1Þu20
3!!!2
pkTSVnoise
where g is the gyromagnetic ratio, (B1/i) is the magnetic field per
unit current generated by the RF coil, VS is the sample volume,
TS is the sample temperature, N is the number of spins, I is the
spin quantum number, u0 is the Larmor precession frequency
(proportional to the static field strength:u0 ¼ gB0), and
Vnoise ¼!!!!!!!!!!!!!!!!!!!!!!!!!4kTRnoiseDf
p
Rnoise is the resistance of the coil plus any other losses due to the
RF circuit and Df is the bandwidth of the receiver. The resistance
in turn is dependent on the size of the coil so that the overall
signal-to-noise for a solenoid coil is proportional to the filling
factor defined as
h ¼ VS
2VC
where VC is the coil volume and the factor of 2 comes from the
fact that for a solenoid coil only half of the B1 field resides within
the coil. For other coil geometries, the filling factor will change
depending on the profile of B1(r).
For microfluidics the filling factor is very small since the
pickup coil must fit around the entire device which is macro-
scopic in size, while the fluid which gives rise to the signal is
microscopic. For conventional NMR the filling factor is close to
50%, while in microfluidics the filling factor can be lower than
10!4, a loss of four orders of magnitude. For NMR which is
already an insensitive technique this hit in signal-to-noise is
devastating.
In order to increase the filling factor several groups have
fabricated planar microcoils directly on the microfluidic
device.14,15 Although less sensitive than solenoid coils, planar
geometries are compatible with planar microfluidic devices and
relatively easy to fabricate. These detectors are significantly more
sensitive than a large, encompassing coil geometry because they
reside very close to the microfluidic channels, increasing the
filling factor, and their small size allows for much higher (B1/i). A
schematic of a microcoil fabricated by micro-photolithography
on top a microfluidic chip is shown in Fig. 1.
Several applications of planar microcoils on lab-on-a-chip
have been demonstrated. Trumbull et al.16 integrated a planar
microcoil on a capillary electrophoresis (CE) chip, demon-
starting a linewidth of less than 1.5 Hz of a 30 nL sample of
water. Wensink et al.15 measured reaction kinetics of imine
formation from benzaldehyde and aniline. Popovic et al.17
recorded spectra of mammalian cells with a sample volume
corresponding to as little as 1800 cells. The main factor that
limits spectral resolution and hence sensitivity is the static
magnetic field inhomogeneities induced by the interfaces of the
coil material, microfluidic components, and fluid inside the
microchannels.14 Although planar microcoil fabrication is
scalable to smaller dimensions, the signal sensitivity due to this
inhomogeneous broadening effect becomes more pronounced.
Furthermore, the sensitivity of the planar microcoils falls off as
r!1, such that path length effects become noticeable, akin to
problems experienced by linear optical techniques. Another
approach demonstrated by Maguire et al.18 utilized microslot
waveguides, which are planar structures based on a dual-layer,
metallic microstrip. These structures are used to transport quasi-
transverse electromagnetic mode (TEM) RF signals on dielectric
materials. This microslot waveguide, due to its geometry does
not have the severe static magnetic field distortions of a planar
microcoil, allowing for higher sensitivity. A two dimensional
COSY spectrum acquired using the microslot probe is shown in
Fig. 2 on ribonuclease-A. While this microslot has not been
integrated directly onto a microfluidic device, in principle such
a detector could be used due to its planar geometry.
Direct imaging
While planar microcoils provide high resolution NMR spectra
directly on the microfluidic device they have two serious draw-
backs that make them less attractive for the purposes of imaging.
First, due to their relatively large fingerprint only a few planar
Fig. 1 Schematic of an electroplated planar microcoil integrated on
a glass microfluidic device with etched channels.
18 | Lab Chip, 2009, 9, 17–23 This journal is ª The Royal Society of Chemistry 2009
structures can be fabricated on a single device.19 Even in the case
of microslots which have a smaller fingerprint and better ability
to pack multiple detectors close together due to weaker inter-
structure couplings, integration of parallel detectors is extremely
difficult due to the complicated electronics that would be
required. Each detector would have to have its own transmitter
and receiver channel which would make the technique extremely
expensive and impractical. For applications involving reactions
or mixing, the chip would have to be designed such that the
interaction of interest occurs precisely in the position of the
detector. Most importantly, the extra expense and complication
of altering already established chip fabrication protocols to
accommodate planar microstructures may offset the potential
benefits of NMR detection in the first place. Although imaging
gradients, could, in principle be used to differentiate channels
that are close in space with planar microcoil detection, this would
negate to a large degree the advantages of using a small detector.
A few groups have recorded images on microfluidic devices by
using large, macroscopic surface coils, even with the poor filling
factor problem. NMR microscopy techniques allow the user to
control the type of flow property to be measured. While MRI
typically detects spin density or relaxation contrast, it is also
possible to encode for velocity, acceleration, or diffusion by
employing multiple gradients that measure the desired phase
while cancelling all other unwanted sources of phase accumula-
tion.20 Ahola et al.21 monitored fluid motion in a micromixer by
measuring the velocity distributions of water at a spatial reso-
lution of 29 mm & 43 mm. Another nice example of this type of
approach is the work by Akpa et al.22 using a conventional
birdcage RF coil, that measured concentration and flow
mapping of immiscible flow inside a low aspect ratio microfluidic
device which would otherwise be difficult to study with optical
techniques that produce en face images. Spin density and velocity
maps through a cross-section of the device are shown in Fig. 3.
Remote detection
A completely different approach to the detection of microfluidic
devices by NMR, pioneered in the lab of Alex Pines at UC
Berkeley, which is known as remote detection takes advantage of
the flow inherent in microfluidics, coupled with the long relaxa-
tion times of the NMR sensor in order to detect the spin
magnetization off the chip with high homogeneity and sensi-
tivity.23,24 By decoupling the excitation and detection of the MR
experiment, it is possible to obtain high sensitivity across the
entire microfluidic device irrespective of the coil filling factor.
The remote detection scheme is shown in Fig. 4A. The fluid
flows through the microfluidic device which is encased inside
a macroscopic RF coil. The nuclear spins of the fluid are initially
Fig. 2 A.Microslot waveguide fabricated with a 248 nm eximer laser. B.
Two-dimensional correlation spectrum (COSY) of ribonuclease-A. Used
with permission from ref. 14.
Fig. 3 Cross-sectional images of immiscible flow through a microfluidic channel of two fluids converging. Spin density and velocity maps of oil (A, C)
and water (B, D), respectively. Used with permission from ref. 18.
This journal is ª The Royal Society of Chemistry 2009 Lab Chip, 2009, 9, 17–23 | 19
excited by application of an RF pulse and begin precession into
the transverse plane. The phase accumulation can proceed by
free evolution (i.e. chemical shift) which encodes spectroscopic
information or in the presence of gradients which can encode
spin density, relaxation weighting, motion, etc. At this point the
encoding scheme is identical to any other pulse sequence avail-
able to NMR or MRI. After adequate phase accumulation the
transverse component of the magnetization is ‘stored’ as longi-
tudinal magnetization by the application of a broadband RF
pulse. This is necessary because the longitudinal relaxation of the
spins (T1) is typically much longer than the transverse relaxation
time (T2). The magnitude of the magnetization along the longi-
tudinal direction is a direct indicator of the phase of the spin at
the moment the ‘storage’ pulse was applied (Fig. 4B). This
encoding typically occurs very rapidly relative to the time scale of
flow such that it can be taken to be instantaneous. The encoded
spins then flow to a highly sensitive microsolenoid detector where
application of a train of hard pulses reads out the magnitude of
the magnetization. Phase cycling is performed to get frequency
discrimination (i.e. quadrature detection) as well as baseline
correction.
Fig. 4C shows an example of ethanol flowing through a single
channel microfluidic device. The spectrum of the ethanol can be
reconstructed by recording the interferogram of chemical-shift
evolution point-by-point and Fourier transforming for each
detection pulse. Here, spatial excitation occurs only over a thin
slice, meaning that this spectrum corresponds to a specific spatial
location on the chip, demonstrating that spectroscopic imaging is
indeed possible (see inset for the full image). In addition, to this
type of image and spectroscopic reconstruction, remote detection
naturally allows the dynamics of the flow to be recorded. Since
the detector is typically much smaller than the encoded volume, it
takes many separate detection acquisitions to record the entire
encoded fluid packet. Since the timing of these pulses is accu-
rately controlled, the time-of-flight (TOF) of the encoded spins
can be mapped and an image or spectrum can be formed for each
detection pulse. Furthermore, because detection occurs outside
the microfluidic device, it is possible to record high resolution
spectra in the detector as well, so that even if the homogeneity on
the microfluidic chip is too poor to perform high resolution
spectroscopy, it is still possible to recover the dynamics of flow
for each species. An example of this concept is illustrated in
Fig. 5A where the confluence of water and ethanol is imaged
through a T-chip device at high spatiotemporal resolution.25 It is
also possible to zoom in on regions of interest by using spatially
selective pulses. This shows that the ethanol and water in fact do
not mix at the low Reynolds used here for the current mixer
geometry.
Fig. 4 Remote detection method: A. Spins are excited by an application of a spatially selective RF pulse (green stripe) in the encoding region (grey
stripes). The magnetization encodes spectroscopic or imaging information in the form of a complex phase which is ‘stored’ by application of
a broadband RF pulse. B. Each phase incrimination corresponds to one point in the indirect interferogram. C. Upon Fourier transformation the
spectrum or image (inset) is formed for each detection pulse.
20 | Lab Chip, 2009, 9, 17–23 This journal is ª The Royal Society of Chemistry 2009
It is also possible to combine imaging and spectroscopy in the
detector as well as to obtain even higher temporal resolution as
shown in Fig. 6.26 This is made possible by recognizing that the
spatial dimension in the detection region is related to TOF of
encoded spins. By slicing up space, one effectively slices up the
time of arrival of the spin packets to a degree determined by the
spatial resolution in the detector. Typically, the residence time in
the detector determines both the spectral resolution as well as the
TOF resolution. However, by recording a spectroscopic image in
the detector it is possible to decouple these two dimensions of the
experiment, bypassing the common assumption that the time
scale of observation of the time variable limits the certainty with
which one can measure the spectral dimension. By employing
spectroscopic imaging in the detector it is possible to overcome
this apparent limit inherent in Fourier pairs, allowing, in prin-
ciple, for arbitrarily high temporal resolution. The enhancement
is evidenced by recording chemically resolved fluid mixing of
benzene and acetonitrile at 500 frames per second (2 ms time
resolution), the highest recorded in a magnetic resonance
imaging experiment.
Comparison of direct and remote detection
The advantages of remote detection are that any microfluidic
device can be used as long as it can fit inside the bore of a high-
field magnet and as long as fluidic components are compatible
with high magnetic fields. However, even in special cases where
these conditions are not met it is possible to perform the remote
detection experiments at low field and even without inductive
detection as shown by Xu et al. who used an optical magne-
tometer to image the flow of water through a phantom.27 The
main advantage is that encoding and detection can be indepen-
dently optimized. Detection proceeds with a very high filling
factor and sensitivity and in a high-homogeneity environment
allowing imaging and spectroscopy at spatial and spectral reso-
lutions difficult to achieve with direct detection methods. The
main disadvantage of remote detection is the need to perform the
experiment in an indirect fashion, causing the total experiment
time to be relatively long. However, compared to direct detection
which requires signal averaging, this does not scale as poorly as
expected. At concentrations of chemical and biological relevance
it is unlikely that direct detection can access the small dimensions
of microfluidic devices for imaging purposes. However, for
spectroscopy, in particular of non-mobile samples such as cells,
direct detection is highly advantageous.
Direct detection can also complement remote detection in
several ways. For example, the ability to record an image both
directly and remotely can give insight into stagnant or recircu-
lating flow. It may also be possible, by placing planar microcoils
at the inlets or outlets of the microfluidic device, to label spins
with significantly improved efficiency. While labelling spins by
a combination of magnetic field gradients and RF pulses is
flexible it does have certain practical disadvantages owing to the
relatively long pulse durations of large macroscopic, and hence
low (B1/i) RF coils. Labelling spins by inversion or saturation
using small planar structures could provide for much faster
encoding and attenuation of flow artefacts. Current research
along both avenues is proceeding rapidly and the field of NMR
on a chip is only a couple of years old. With the advent of
commercial hyperpolarization methods it should be possible to
substantially increase the sensitivity of both direct and remote
detection methods.28
Fig. 5 Schematic of time-of-flight (TOF) imaging of two fluids inside a T-mixing chip (A) based on chemical shift selection in the detector (B). For each
detection pulse an image is formed of each species separated by their chemical shift (C).
This journal is ª The Royal Society of Chemistry 2009 Lab Chip, 2009, 9, 17–23 | 21
Conclusions
Detection by NMR opens up the possibility of using a whole new
class of materials for microfluidic device fabrication as optical
transparency is no longer required. No tracers are used in NMR
and due to the noninvasive nature of magnetic resonance, there is
never the worry that the hydrodynamic properties of the fluid are
somehow altered. While the spatial resolution of magnetic
resonance is below that of optical detection, it should be possible
to obtain better than 5 mm isotropic resolution which is adequate
for most applications. Furthermore, the use of relaxation
contrast (i.e. T1 and T2 weighting) and chemical shift allows
differentiation of fluids irrespective of their optical properties. By
employing three-dimensional imaging methods, any point on the
microfluidic device can be analyzed, unlike most optical methods
that are restricted to the en face type where the concentration
profiles are a projection through the entire depth of the channel.
The ability to easily manipulate the spins by application of RF
fields allows for a large body of information to be recorded,
whether it be fluid mechanics (e.g. velocity, acceleration,
diffusion) or chemical and physical properties (e.g. chemical
shift, scalar couplings, correlation spectroscopy, reaction
kinetics, dynamics). Future work along the lines of exploiting
many of the tools available to MR towards lab-on-a-chip
applications promises to be an exciting area of research and
development.
Acknowledgements
Discussions, encouragement, and support from Professor Alex
Pines is greatly appreciated.
Fig. 6 A. Schematic of remote detection with time slicing of the TOF dimension. A spectroscopic image is formed in the detector, with each position
along the 1D profile corresponding to a different TOF value of encoded spins. B. Partial images taken from the integrated data set. For each point in the
image a TOF curve is measured which gives information about the time of arrival and dispersion of the encoded fluid voxel. Differences at the outlet
(green curve) show that the fluid species begin to separate in the dead volume near the outlet connector. C. Comparison of spectra acquired inside and
outside of the chip showing that the resolution off the chip is dramatically improved compared to on the chip.
22 | Lab Chip, 2009, 9, 17–23 This journal is ª The Royal Society of Chemistry 2009
References
1 S. Devasebathipathy, J. G. Santiago, S. T. Wereley, C. D. Meinhartand K. Takehara, Exp. Fluids, 2001, 34, 504.
2 D. Ross and L. E. Locascio, Anal. Chem., 2003, 75, 1218.3 S.-A. Leung, R. F. Winkle, R. C. R. Wootton and A. J. de Mello,Analyst, 2005, 5, 431.
4 F. Sarrazin and J.-B. Salmon, Anal. Chem., 2008, 80, 1689.5 H. Rampel and J. M. Pope, Conc. Magn. Reson., 1993, 5, 43.6 H. Rampel and J. M. Pope, Conc. Magn. Reson., 1993, 5, 43.7 K. Wuthrich, NMR of Proteins and Nucleic Acids, New York, Wiley,1986.
8 E. MacNamara, T. Hou, G. Fisher, S. Williams and D. Raftery,Anal.Chim. Acta, 1999, 397, 9.
9 D. L. Olson, T. L. Peck, A. G. Webb, R. L. Magin and J. V. Sweedler,Science, 1995, 270, 5244.
10 J. A. Rogers, R. J. Jackman, G. M. Whitesides, D. L. Olson andJ. V. Sweedler, Appl. Phys. Lett., 1997, 70, 2464.
11 D. Rugar, R. Budakian, H. J. Mamin and B. W. Chui, Nature, 2004,430, 329.
12 A. Abragham, Principles of Nuclear Magnetism, Oxford, OxfordUnivesity Press, 1961.
13 D. I. Hoult and R. E. Richards, J. Magn. Reson., 1976, 24, 71.14 C. Massin, F. Vincent, A. Homsy, K. Ehrmann, G. Boero,
P.-A. Besse, A. Daridon, E. Verpoorte, N. F. de Rooij andR. S. Popovic, J. Magn. Reson., 2003, 164, 242.
15 H. Wensink, F. Benito-Lopez, D. C. Hermes, W. Verboom,H. J. G. E. Gardeniers, D. N. Reinhoudt and A. Van Den Berg,Lab Chip, 2005, 5, 280.
16 J. D. Trumbull, I. K. Glasgow, D. J. Beebe and R. L. Magin, IEEETrans. Biomed. Eng., 2000, 47, 3.
17 K. Ehrmann, K. Pataky, M. Stettler, F. M. Wurm, J. Brugger,P.-A. Besse and R. Popovic, Lab Chip, 2007, 7, 381.
18 Y. Maguire, I. L. Chuang, S. Zhang and N. Gershenfeld, Proc. Natl.Acad. Sci. U. S. A., 2007, 104, 9198.
19 K. Ehrmann, M. Gersbach, P. Pascoal, F. Vincent, C. Massin,D. Stamou, P.-A. Besse, H. Vogul and R. S. Popovic, J. Magn.Reson., 2006, 178, 96.
20 P. T. Callaghan, Principles of Nuclear Magnetic ResonanceMicroscopy, Oxford University Press, Oxford, 1991.
21 S. Ahola, F. Casanova, J. Perlo, K. Munnemann, B. Blumich andS. Stapf, Lab Chip, 2006, 6, 90.
22 B. S. Akpa, S. M. Matthews, A. J. Sederman, K. Yunus, A. C. Fisher,M. L. Johns and L. F. Gladden, Anal. Chem., 2007, 79,6128.
23 A. J. Moule, M. M. Spence, S. I. Hans, J. A. Seeley, K. L. Pierce,S. Saxena and A. Pines, Proc. Natl. Acad. Sci. USA, 2003, 100,9122.
24 J. Granwehr, E. Harel, S. Hans, S. Garcia, A. Pines, P. N. Sen andY. Q. Song, Phys. Rev. Lett., 2005, 95, 075503.
25 E. Harel, C. Hilty, K. Koen, E. E. McDonnell and A. Pines, Phys.Rev. Lett., 2007, 98, 017601.
26 E. Harel and A. Pines, J. Magn. Reson., 2008, 193, 199–206.27 S. Xu, V. V. Yashchuk, M. H. Donaldson, S. M. Rochester,
D. Budker and A. Pines, Proc. Natl. Acad. Sci. U. S. A., 2006, 103,12668.
28 L. Becerra, G. J. Gerfen, R. J. Temkin, D. J. Singel and R. G. Griffin,Phys. Rev. Lett., 1993, 71, 5361.
This journal is ª The Royal Society of Chemistry 2009 Lab Chip, 2009, 9, 17–23 | 23